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A tetracycline-regulated reporter system was used to investigate the regulation of cyclooxygenase 2 (Cox-2) mRNA stability by the mitogen-activated protein kinase (MAPK) p38 signaling cascade. The stable ␤-globin mRNA was rendered unstable by insertion of the 2,500-nucleotide Cox-2 3 untranslated region (3 UTR). The chimeric transcript was stabilized by a constitutively active form of MAPK kinase 6, an activator of p38. This stabilization was blocked by SB203580, an inhibitor of p38, and by two different dominant negative forms of MAPK-activated protein kinase 2 (MAPKAPK-2), a kinase lying downstream of p38. Constitutively active MAPKAPK-2 was also able to stabilize chimeric ␤-globin-Cox-2 transcripts. The MAPKAPK-2 substrate hsp27 may be involved in stabilization, as ␤-globin-Cox-2 transcripts were partially stabilized by phosphomimetic mutant forms of hsp27. A short (123-nucleotide) fragment of the Cox-2 3 UTR was necessary and sufficient for the regulation of mRNA stability by the p38 cascade and interacted with a HeLa protein immunologically related to AU-rich element/poly(U) binding factor 1.

p38 mitogen-activated protein kinase (MAPK) is activated by inflammatory stimuli such as bacterial lipopolysaccharide (LPS), interleukin-1, and tumor necrosis factor. We have previously shown that the pyridinyl imidazole SB 203580, which inhibits it, blocks the interleukin-1 induction of cyclooxygenase-2 (COX-2) and matrix metalloproteinase 1 and 3 mRNAs in fibroblasts. Here we explore the role of p38 MAPK in the response of human monocytes to LPS. 0.1 M SB 203580 significantly inhibited the LPS induction of COX-2 and tumor necrosis factor protein and mRNAs. The activity of MAPKactivated protein kinase-2 (a substrate of p38 MAPK) in the cells was commensurately reduced. Some isoforms of c-jun N-terminal kinase (which is also activated by LPS) are sensitive to SB 203580; the inhibitor had little effect on monocyte c-jun N-terminal kinases up to 2 M. We investigated the mechanism of inhibition of COX-2 induction. Transcription (measured by a nuclear run-on assay) was 60% inhibited by SB 203580 (2 M). Importantly, we found that p38 MAPK was essential for stabilizing COX-2 mRNA: when cells stimulated for 4 h with LPS were treated with actinomycin D, COX-2 mRNA decayed slowly. Treatment of stimulated cells with 2 M SB 203580 caused a rapid disappearance of COX-2 mRNA, even with actinomycin D present. We conclude p38 MAPK plays a role in the transcription and stabilization of COX-2 mRNA.p38 mitogen-activated protein kinase (MAPK) 1 is a member of the MAPK family and is activated by the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF), by bacterial lipopolysaccharide (LPS), and by a range of cellular stresses (1-5). Although originally characterized as a stress or inflammatory kinase, it is likely to have diverse functions because it is also activated in platelets by thrombin and collagen (6) and in T cells upon activation by various stimuli (7) and is constitutively active in liver (8, 9). Little is known about the physiological functions it controls. One substrate is MAPKactivated protein kinase-2 (MAPKAPK-2) (10, 11), which in turn phosphorylates the small heat shock protein hsp27 (12) and the cAMP-response element binding protein (13). Other putative targets are the MAPK integrating kinase (14, 15) and the transcription factors CHOP (16), myocyte enhancer factor 2C (17), and activating transcription factor 2 (4).Besides the original p38 MAPK (called ␣), a closely similar ␤ form has been described (18) as well as two more distantly related enzymes that also contain the TGY motif: stress-activated protein kinase 3 (or p38␥) (19 -21) and stress-activated protein kinase 4 (or p38␦) (22-24). The p38␣ and p38␤ MAPKs are inhibited by a class of pyridinyl imidazole compounds of which the best characterized is SB 203580 (11). These were first identified as inhibitors of TNF (and IL-1) production by LPS-activated monocytes (25) and were later shown to inhibit p38 MAPK (5, 11). The pyridinyl imidazoles inhibited TNF (and IL-1) protein production with relatively little effect on the levels of mRNA ...

During inflammatory reactions, activated leukocytes are thought to produce a variety of small proteins (cytokines) that influence the behaviour of other cells (including other leukocytes). Of these substances, which include the interleukins, interferons and tumour necrosis factors (TNFs), interleukin-1 (IL-1) has been considered potentially a most important inflammatory mediator because of its wide range of effects. In vivo it is pyrogenic and promotes the acute phase response; in vitro it activates lymphocytes and stimulates resorption of cartilage and bone. Cartilage resorption is a major feature of inflammatory diseases such as rheumatoid arthritis, and IL-1 is the only cytokine hitherto known to promote it. TNFs are characterized by their effects on tumours and cytotoxicity to transformed cells, but share some actions with IL-1. I report here that recombinant human TNF alpha stimulates resorption and inhibits synthesis of proteoglycan in explants of cartilage. Its action is similar to and additive with IL-1, and it is a second macrophage-derived cytokine whose production in rheumatoid arthritis, or inflammation generally, could contribute to tissue destruction.

Glucocorticoids (GCs), which are used in the treatment of immune-mediated inflammatory diseases, inhibit the expression of many inflammatory mediators. They can also induce the expression of dual specificity phosphatase 1 (DUSP1; otherwise known as mitogen-activated protein kinase [MAPK] phosphatase 1), which dephosphorylates and inactivates MAPKs. We investigated the role of DUSP1 in the antiinflammatory action of the GC dexamethasone (Dex). Dex-mediated inhibition of c-Jun N-terminal kinase and p38 MAPK was abrogated in DUSP1−/− mouse macrophages. Dex-mediated suppression of several proinflammatory genes (including tumor necrosis factor, cyclooxygenase 2, and interleukin 1α and 1β) was impaired in DUSP1−/− mouse macrophages, whereas other proinflammatory genes were inhibited by Dex in a DUSP1-independent manner. In vivo antiinflammatory effects of Dex on zymosan-induced inflammation were impaired in DUSP1−/− mice. Therefore, the expression of DUSP1 is required for the inhibition of proinflammatory signaling pathways by Dex in mouse macrophages. Furthermore, DUSP1 contributes to the antiinflammatory effects of Dex in vitro and in vivo.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway, acting through the downstream kinase MK2, regulates the stability of many proinflammatory mRNAs that contain adenosine/uridine-rich elements (AREs). It is thought to do this by modulating the expression or activity of ARE-binding proteins that regulate mRNA turnover. MK2 phosphorylates the ARE-binding and mRNA-destabilizing protein tristetraprolin (TTP) at serines 52 and 178. Here we show that the p38 MAPK pathway regulates the subcellular localization and stability of TTP protein. A p38 MAPK inhibitor causes rapid dephosphorylation of TTP, relocalization from the cytoplasm to the nucleus, and degradation by the 20S/26S proteasome. Hence, continuous activity of the p38 MAPK pathway is required to maintain the phosphorylation status, cytoplasmic localization, and stability of TTP protein. The regulation of both subcellular localization and protein stability is dependent on MK2 and on the integrity of serines 52 and 178. Furthermore, the extracellular signal-regulated kinase (ERK) pathway synergizes with the p38 MAPK pathway to regulate both stability and localization of TTP. This effect is independent of kinases that are known to be synergistically activated by ERK and p38 MAPK. We present a model for the actions of TTP and the p38 MAPK pathway during distinct phases of the inflammatory response.The tandem zinc finger protein tristetraprolin (TTP; also known as Nup475, Tis11, or Zfp36) (23,26,40,46,62) is expressed in activated monocytic cells (13, 47) and T lymphocytes (49, 51). It functions to regulate the expression of tumor necrosis factor ␣ (TNF-␣) by binding to a conserved adenosine/uridine-rich element (ARE) within the 3Ј-untranslated region of TNF-␣ mRNA (13,31,32,36,47). TTP promotes both mRNA deadenylation and 3Ј to 5Ј degradation of the mRNA body (35, 37-39), consistent with its ability to recruit several factors involved in these processes (14,25,39,45). The pivotal role of TTP in the regulation of TNF-␣ is illustrated by the proinflammatory phenotype of a TTP Ϫ/Ϫ mouse strain, in which chronic overexpression of TNF-␣ by macrophages results in severe polyarthritis and cachexia (11,13,57). TTP has also been implicated in the posttranscriptional regulation of granulocyte-macrophage colony-stimulating factor (12), interleukin-2 (51), cyclooxygenase 2 (COX-2) (50), and inducible nitric oxide synthase (24). It may also regulate its own expression by binding to an ARE in the 3Ј untranslated region of TTP mRNA (60). The minimum binding site of TTP is the nonameric sequence UUAUUUAUU (2,3,38,65), and it is likely that additional posttranscriptional targets of TTP containing this sequence remain to be identified.The p38 mitogen-activated protein kinase (MAPK) and its downstream kinase MK2 play a central role in the posttranscriptional regulation of inflammatory gene expression in myeloid and other cells (5, 16, 20-22, 33, 34, 54). We and others have therefore investigated interactions of the p38 MAPK pathway with TTP. In a mouse macrophage-like...

The stress-activated protein kinase p38 stabilizes a number of mRNAs encoding inflammatory mediators, such as cyclooxygenase 2 (Cox-2). In HeLa cells the anti-inflammatory glucocorticoid dexamethasone destabilizes Cox-2 mRNA by inhibiting p38 function. Here we demonstrate that this effect is phosphatase dependent.